Vaccinex has pioneered the use of recombinant cDNA libraries constructed in a vaccinia virus based vector for the identification of tumor antigens (Nature Medicine 7:967-972, 2001). More recently, Vaccinex has extended use of the vaccinia library technology to select high-affinity, fully human monoclonal antibodies from immunoglobulin gene libraries expressed in mammalian cells. In these different applications, the selection of specific recombinants from libraries constructed in vaccinia virus has proven to be extremely efficient. This is in large measure due to the ease with which recombinant virus can be recovered from even a very few selected cells - perhaps as few as only one infected cell. We propose here a novel application of the vaccinia library technology toward the functional selection of new targets for cancer therapy. Specifically, beginning with a marker gene known to be relevant to cancer, this strategy will efficiently identify other genes that regulate expression of this marker gene and that may, therefore, also be relevant to tumorigenesis. We focus in this proposal on C35, a novel marker of breast cancer first identified in our laboratory. C35 is highly overexpressed in 70% of primary and metastatic human breast tumors and 30% of bladder carcinomas but is not detected at the protein level in any of 31 different normal tissues except in the Leydig cells of testes. Our strategy is to introduce into a low C35 expressing cell line a reporter construct in which the identified minimal C35 promoter drives expression of green fluorescent protein. These cells will be infected with a vaccinia virus cDNA library constructed from a high-C35 expressing cell line. Any vaccinia virus recombinants encoding genes capable of activating the C35 promoter (and therefore the reporter) will be efficiently selected by FACS. Identification of transcription factors and other upstream regulators of C35 expression will elucidate novel regulatory relationships and, most importantly from a commercial perspective, will expand the repertoire of therapeutic targets and modes of intervention in cancer.